Hampton M L, Freese E
J Bacteriol. 1974 May;118(2):497-504. doi: 10.1128/jb.118.2.497-504.1974.
Lineweaver-Burk plots of reduced nicotinamide adenine dinucleotide (NADH) oxidation by membrane preparations from Bacillus subtilis are biphasic, with two K(m) values for NADH. The higher K(m) corresponds to the only K(m) observed for NADH oxidation by whole cells, whereas the lower K(m) corresponds to that observed with open cell envelopes. Membrane preparations apparently contain a small fraction of open or inverted vesicles which is responsible for the low K(m) reaction, whereas entry of NADH into the larger portion of closed, normally oriented vesicles is rate limiting and responsible for the high K(m) reaction. In contrast, the oxidation of l-alpha-glycerol-phosphate (glycerol-P) by membrane preparations shows only one K(m) that corresponds to that of glycerol-P oxidation by whole cells or lysates. Since glycerol-P dehydrogenase (NAD independent) has the same K(m), this enzyme reaction rather than entry of glycerol-P into vesicles represents the rate-limiting step for glycerol-phosphate oxidation. The K(m) for amino acid uptake by vesicles in the presence of NADH corresponds to the high K(m) for NADH oxidation, indicating that NADH energizes transport only if it enters closed, normally oriented vesicles. Studies with rotenone and proteolytic enzymes support this interpretation. The apparent efficiency of NADH in energizing uptake seems to be lower than that of glycerol-P because, under the experimental conditions usually employed, open or inverted vesicles that do not participate in amino acid uptake are responsible for the major portion of NADH oxidation. When the results are corrected for this effect, the efficiency of NADH is essentially the same as that of l-alpha-glycerol-P.
枯草芽孢杆菌膜制剂对还原型烟酰胺腺嘌呤二核苷酸（NADH）氧化的Lineweaver-Burk图呈双相，NADH有两个米氏常数（K(m)）值。较高的K(m)值对应于全细胞NADH氧化时观察到的唯一K(m)值，而较低的K(m)值对应于开放细胞包膜时观察到的K(m)值。膜制剂显然含有一小部分开放或倒置的囊泡，它们负责低K(m)反应，而NADH进入较大部分封闭的、正常取向的囊泡是限速步骤，负责高K(m)反应。相比之下，膜制剂对L-α-甘油磷酸（甘油-P）的氧化仅显示一个K(m)值，该值与全细胞或裂解物对甘油-P的氧化K(m)值相对应。由于甘油-P脱氢酶（不依赖NAD）具有相同的K(m)，该酶反应而非甘油-P进入囊泡代表甘油磷酸氧化的限速步骤。在有NADH存在的情况下，囊泡对氨基酸摄取的K(m)值对应于NADH氧化的高K(m)值，表明只有当NADH进入封闭的、正常取向的囊泡时，它才为转运提供能量。用鱼藤酮和蛋白水解酶进行的研究支持这一解释。NADH在为摄取提供能量方面的表观效率似乎低于甘油-P，因为在通常采用的实验条件下，不参与氨基酸摄取的开放或倒置囊泡负责NADH氧化的大部分。当对这种效应进行校正后，NADH的效率与L-α-甘油-P的效率基本相同。
